Reverse Genetics Flashcards
What does reverse genetics seek to find?
phenotypes liked to specific sequences of DNA
deleting, disrupting or producing mutations in specific gene may reveal phenotypes that give clues to functions
What are the three steps in reverse genetics?
- Alter gene in vitro
- Introduce into cell
- Determine phenotypic effects
What are the different ways to knockout a gene? (5)
- Gene knockout
- replace with expressed in wrong tissue
- certain genes deleted in certain tissues
- slight changes in protein structure
- observing the effects of not having the genes
How do you alter a gene in vitro?
string nucleotides together in lab
create DNA sequence
use recombinant DNA techniques
- design a primer
- extension step happens
- included mutation
go through multiple cycles, plasmid created as PCR product
How do you introduce DNA into cell?
Take in plasmid through pores
transfection not transformation in animals
direct uptake of DNA:
incubate DNA with component cells
- bacterial/yeast transformation
- animal cell transfection
methods to induce DNA uptake:
- electroporation
- microinjection
- virus-mediated
- ballistic (gene gun)
fate of the transgene:
- transient expression
- replaces on a plasmid
- chromosomal integration
How does transient transfection work?
promotor sequence will be expressed
eventually plasmid sequence will break down unlike Bacteria
How do you determine phenotypic effects?
not unusual for no effect
can see the wild type then compare with symptoms on mutant e.g. premature ageing
What is gene targeting?
transgenic organisms = genetically engineered organisms
transgenes = modified genes
DNA carrying mutated gene in cell, inserts into genome by homologous recombination
specific genes can be targeted by gene crossing over
create unique barcode sequences
then undergo homologous recombination and create a knockout
Targeting deletion of diploid yeast requires two marker genes
knock out both copies
introduce disruption cassette can undergo homologous recombination
targeted the gene and introduce to different genes on different strands
identify to show what copies have been knocked out
What is the marker recycling scheme?
using Credit/Lox recombination system
knocked out target gene but also removed the markers
can then go and knock out another gene
knock out multiple different strains
Targeted gene deletion or replacement of diploid animal
more complex
lines of mice with functional gene altered/removed called knockout mice
used to determine function of many mice genes
put targeted gene in embryonic stem cells
inject & will get different mice from hybridised embryos
What is CRISPR?
first coined in 2002
Clustered Regularly Interspaced Short Palindromic Repeats
high likelihood for mutation in the gene, usually non functional
What is step 1 in CRISPR?
Short viral DNA sequence is integrated into CRISPR locus
What is step 2 in CRISPR?
RNA is transcribed from CRISPR locus, processed and bound to Cas protein
What is step 3 in CRISPR?
Small crRNA in complex with Cas seeks out and destroys viral sequences
when reinfected, the incoming viral DNA is destroyed by the crDNA
